The long-term objective of this research is to develop novel, stem cell-based methods to study and treat hearing loss in humans. Hearing loss is one of the most common disabilities globally, affecting over 275 million people. In most cases of hearing loss, the hair cells of the inner ear have been lost or damaged. These cells cannot regenerate spontaneously, leading to the permanency that typifies hearing loss in most affected individuals. However, mouse pluripotent stem cells can be used to generate functional inner ear hair cell-like cells in vitro. In addition, stem cells survive following transplantation into the rdent cochlea. These two findings have fueled enthusiasm for exploring the use of stem cells as a means to improve our understanding of hearing loss and to treat patients with some forms of deafness. Two immediate challenges in realizing the promise of stem cell-based disease modeling and therapies for hearing loss have become clear. First, a method for the use of human pluripotent stem cells to generate inner ear hair cells has not been identified. Second, stem cells transplanted into the adult mammalian inner ear do not integrate into the auditory sensory epithelium (the organ of Corti) as hair cells. The two specific aims described in this grant directly address these immediate challenges. The studies in Aim 1 build upon preliminary findings that human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can be directed in their differentiation to otic placode progenitor cells using a well-defined neural differentiation paradigm. We propose to optimize our culture conditions for efficient generation of otic progenitor cells and/or enrich this population. We will characterize these cells using PCR, immunohistochemistry and flow cytometry. Aim 2 builds upon findings that mouse pluripotent stem cells integrate into the auditory sensory epithelium and differentiate into hair cells upon transplantation into the developing chick inner ear. We will use in utero transplantation into the mouse otocyst to assess the ability of human iPSC-derived otic progenitor cells to respond to developmental cues and integrate into the organ of Corti as hair cells. We will evaluate the long- term survival, integration and differentiation of grafted cells using immune-histochemistry and electron microscopy. Successful completion of the studies in Aim 1 will provide a platform for the use of ESC and patient- derived, disease-specific iPSC to model hair cell development and disease in the future. Completion of the studies in Aim 2 will provide a novel method for identifying the factors most critical to allow for integration of grafted stem cells into the mammalian inner ear. Together the proposed research will uniquely address two critical challenges in auditory science and form a significant foundation for continued pursuit of our long term goals.